A modified mouse air pouch model for evaluating the effects of compounds on granuloma induced cartilage degradation.

1. Employing rat femoral head cartilage implanted in a 6 day old mouse air pouch, the effects of inflammatory stimuli (i.e. cotton pellets, carrageenan, zymosan) on the loss of proteoglycan and collagen and granuloma formation have been studied. 2. Wrapping of the cartilage in cotton resulted in granuloma formation with accelerated loss of proteoglycan and collagen over the 14 day implantation period. The amount of loss increased with increasing weight of cotton. 3. The effects of different classes of anti-rheumatic drugs on granuloma formation and proteoglycan and collagen loss from cotton wrapped femoral head cartilage in the mouse air pouch have been studied. 4. Non-steroidal anti-inflammatory drugs (NSAIDs) had no influence on granuloma formation, but in general accelerated the rates of proteoglycan and collagen loss. 5. Dexamethasone and prednisolone significantly reduced granuloma formation and had a marked protective effect on cartilage breakdown. 6. Of the slow acting anti-rheumatic drugs examined, only gold sodium thiomalate (GSTM) and dapsone significantly decreased cartilage loss, with an accompanying modest decrease in granuloma formation. 7. The immunosuppressants cyclophosphamide and methotrexate, but not azathioprine, reduced cartilage degradation, but had no effect on granuloma formation. 8. The results for the different classes of anti-inflammatory and anti-rheumatic drugs are discussed in relation to their effects in other animal models and their reported therapeutic activities in man. It is concluded that the mouse air pouch method as described offers advantages as an animal model over existing procedures to predict therapeutic efficacy in man.

Proteoglycan biosynthesis as a determinant of patella damage in the murine antigen-induced arthritis model.

The incorporation of 35SO4 into cartilage proteoglycan has been employed as a measure of patella damage in the murine antigen-induced arthritis model. In a preliminary set of experiments, where both the proteoglycan concentration and 35SO4 incorporation were determined in control and arthritic patella over a 2 day to 6 day week period, the arthritic joint contained significantly higher levels of radioactivity compared with the controls. A subsequent study over an extended period of 10 weeks confirmed the earlier results, and indicated that the 6 week samples showed the greatest difference (71%) in 35SO4 incorporation between the arthritic and control patella.

The serum amyloid P response in the mouse air pouch.

Levels of the acute phase reactant serum amyloid P (SAP) have been measured in the mouse pouch model of rheumatoid arthritis. Implantation of cartilage resulted in a significant and rapid elevation in the SAP concentration, which remained high for the duration of the experiment (14 days). Initial studies with several clinically employed antirheumatic drugs indicated that dexamethasone and cyclosporin A had a marked inhibitory effect.

Antisecretory effects of two new histamine H2-receptor antagonists.

N-(3-[3-(1-Piperidinylmethyl)phenoxy]propyl)acetoxyaceta mide hydrochloride (Hoe 760) and N-(3-[3-(1-piperidinylmethyl)phenoxy]propyl)glycolamine hydrochloride (Hoe 062) are highly specific H2-receptor antagonists. The compounds are equipotent after intragastrical or intravenous administration. The antagonists inhibited gastric acid secretion in the rat induced by all stimuli tested, carbachol, desglugastrin and histamine. In the Heidenhain pouch dog whose gastric acid secretion was stimulated by food or histamine the two receptor blockers proved to be 4-6 times more potent inhibitors than cimetidine.

The determination of receptor constants for histamine H2-agonists in the guinea-pig isolated right atrium using an irreversible H2-antagonist.

From measurements of chronotropy in the guinea-pig isolated right atrium, a compound (E1309) was found which behaved as an irreversible antagonist at the histamine H2 receptor. E1309 was used to block irreversibly a proportion of the H2 receptors and the dissociation constants, relative efficacies and receptor reserves of four H2-agonists were determined. The calculated dissociation constants were similar to the Ki values reported from H2-radioligand binding studies but different from the observed EC50 values. The order of potency for the four H2-agonists was impromidine much greater than histamine greater than dimaprit greater than 4-methylhistamine. The order of relative efficacy was 4-methylhistamine greater than dimaprit greater than histamine greater than impromidine, the natural agonist not being the most efficacious. This atypical finding is discussed in relation to other receptor classes.

A study of the H2-receptor for histamine stimulating adenylate cyclase in homogenates of guinea-pig lung parenchyma.

The effect of forskolin and several H2-agonists was investigated on the activity of adenylate cyclase in homogenates of guinea-pig lung parenchyma. Histamine, 0.1 microM to 1 mM, dimaprit, 1 microM to 10 mM, 4-methyl histamine, 0.1 microM to 10 mM, impromidine, 10 nM to 10 microM and forskolin, 1 nM to 100 microM, all produced a dose-dependent stimulation of adenylate cyclase activity above the basal level. The histamine H1-receptor antagonist mepyramine, 10 microM, and beta-adrenoceptor antagonist propranolol, 10 microM, had no effect on the stimulation by histamine of adenylate cyclase. The dose-response curve for stimulation by histamine of adenylate cyclase was shifted to the right in a dose-dependent manner by increasing concentrations of several H2-antagonists. Schild plots constructed for each H2-antagonist produced straight lines with slopes not significantly different from unity. The equilibrium dissociation constants obtained for the H2-antagonists in this study were similar to those previously reported for inhibition of dimaprit-induced relaxation of the pre-contracted lung strip, inhibition of [3H]-tiotidine binding to homogenates of guinea-pig lung parenchyma and inhibition of histamine-stimulated adenylate cyclase in guinea-pig gastric mucosa.

In vitro histamine H2-antagonist activity of the novel compound HUK 978.

Histamine stimulated adenylate cyclase from guinea-pig fundic mucosa and 3H-tiotidine binding in guinea-pig cerebral cortex were used to assess the in-vitro histamine H2-activity of the novel H2-antagonist HUK 978. The results showed that HUK 978 was a more potent H2-antagonist than either cimetidine or ranitidine. HUK 978 was also shown to be devoid of activity at the histamine H1-receptor, the muscarinic receptor and the alpha and beta-adrenergic receptors.

Anti-secretory activity of the novel H2-antagonist, HUK 978, in rat, guinea-pig and dog.

The anti-secretory activity of the competitive H2-antagonist HUK 978 was determined in rat, guinea-pig and dog. In all systems examined, HUK 978 was more potent than cimetidine and ranitidine both intravenously and orally. In addition, the compound at approximately equipotent doses as these established H2-antagonists exhibited a significantly longer inhibitory profile following oral and systemic administration. Data from these pharmacological studies and the in vitro investigations previously reported, suggest that HUK 978 is a highly specific H2-antagonist and inhibits acid secretion for longer periods than other competitive compounds.

A study of the histamine H2-receptor mediating relaxation of the parenchymal lung strip preparation of the guinea-pig.

The relaxation produced by several H2-receptor agonists and forskolin was investigated on strips of guinea-pig lung parenchyma. Dimparit, 1 microM to 10 mM, 4-methyl histamine, 0.5 microM to 100 microM and impromidine, 10 nM to 1 microM, had no effect on the tone of the unstimulated strips of lung parenchyma but caused a dose-dependent relaxation of strips that were contracted by 2-pyridylethylamine (2-PEA), 15 microM. Forskolin, 10 nM to 4 microM, produced a dose-dependent relaxation of both the stimulated and unstimulated lung strips. The muscarinic antagonist atropine, 1 microM, and the beta 2-adrenoceptor antagonist propranolol, 10 microM, had no effect on the dose-response curve for dimaprit-induced relaxation of the lung strip. The dose-response curve for dimaprit was shifted to the right in a dose-dependent manner by increasing concentrations of a variety of H2-antagonists. Schild plots produced a straight line for all the H2-antagonists with slopes not significantly different from unity. The equilibrium dissociation constants for the H2-antagonists on the lung strip preparation were similar to those previously reported for inhibition of the chronotropic activity of histamine on guinea-pig right atria and inhibition of [3H]-tiotidine binding to homogenates of guinea-pig lung parenchyma.

The binding of [3H]-tiotidine to homogenates of guinea-pig lung parenchyma.

By use of a rapid filtration assay, the binding of [3H]-tiotidine to homogenates of guinea-pig lung parenchyma was found to be saturable and of a high affinity. Mean values for the KD and Bmax were calculated as 8.5 +/- 1.5 nM and 28 +/- 5 fmol mg-1 protein respectively. The association and dissociation rate constants for [3H]-tiotidine binding at 4 degrees C were calculated to be 0.81 +/- 0.06 microM min-1 and 0.063 +/- 0.005 min-1 respectively, yielding a kinetically derived KD of 7.8 nM. A wide range of H2-receptor agonist and antagonists displaced [3H]-tiotidine binding from lung parenchyma homogenates in a biphasic manner. Examination of the first phase of the displacement of [3H]-tiotidine yielded Ki values for the antagonists tested similar to those found in other binding studies using this ligand and similar to KB values calculated for the antagonists in pharmacological studies.

The use of an in vitro binding assay to predict histamine H2-antagonist activity.

Thirty-seven compounds were evaluated for their ability to inhibit histamine stimulated adenylate cyclase and to inhibit 3H-tiotidine binding. The compounds examined included a number of known H2-antagonists and a number of potential H2-antagonists of diverse chemical structure. The correlation between the Ki values from the binding assay and from the inhibition of adenylate cyclase was calculated to be r = 0.99, p less than 0.001. Thus 3H-tiotidine binding in guinea-pig cerebral cortex can be used to give a valid assessment of histamine H2-receptor activity.

Low level antigen responses in guinea-pig lung strip.

Guinea-pigs were sensitized either by i.p. administration or by a novel procedure involving inhalation of ovalbumin (2, 0.2 and 0.002%) on days 0, 5 and 19 respectively. Lung strips from these guinea-pigs were challenged with both low (0.02 micrograms/ml) and high (10 micrograms/ml) concentrations of ovalbumin and the responses compared. Whereas the low level antigen gave consistent contractions following aerosol sensitization, no response was observed from the i.p. sensitized guinea-pig lung strips. Marked differences were also observed following the high ovalbumin challenge, where the aerosol sensitized lungs gave almost twice the response as tissue from the i.p. sensitized guinea-pigs, the former being approximately 140% of that observed with acetyl-beta-methyl choline (1 mM). Furthermore, the response elicited in the lungs from aerosol sensitized guinea-pigs were not modified by the addition of high concentrations of the H1-antagonist diphenhydramine (100 microM), before or subsequent to challenge. The data suggest that the aerosol sensitization procedure gives rise to a contractile response in guinea-pig lung strips which contains no observable histamine component.

Histamine H2-binding studies in the guinea-pig brain.

Using 3H-mepyramine and 3H-tiotidine to label, respectively, the histamine H1- and H2-receptors, we have assessed the distribution of these receptors in five distinct areas of guinea-pig brain. With regard to the H2-receptor, we could detect no binding in either the cerebellum or pons-medulla, whereas the distribution in the other brain areas was corpus striatum greater than cortex greater than hippocampus. This was in contrast to the distribution of the H1-receptor which was found to be in all areas studied but predominantly in the cerebellum.

A new in vivo method for the measurement of repetitive anaphylactic responses in the guinea-pig.

The established Konzett-Rossler bronchorespiratory model has been combined with a unique ovalbumin sensitization procedure to give a novel method to measure anaphylaxis in the anaesthetized guinea-pig. Following antigen challenge, up to eight equal bronchoconstrictor responses to the same dose can be generated from a single animal over a 120 min period. Total inhibition of the anaphylactic response can be elicited by four different classes of compound, namely salbutamol, mepyramine, theophylline and dimaprit. Cromoglycate failed to cause any inhibition. The method is discussed with particular reference to the antigen sensitization procedure, which differs substantially from other regimens previously employed and gives rise to heat labile antibody.

Specific binding of 3H-tiotidine to histamine H2 receptors in guinea pig cerebral cortex.

Although the H2 subclass of histamine receptor has been revealed by classical pharmacological approaches, the direct identification of this adenylate cyclase-linked receptor has, despite much effort, remained elusive. Initial studies using 3H-metiamide and 3H-histamine and, subsequently, work from our own laboratory and others using 3H-cimetidine and 3H-ranitidine in various tissues, has shown the unsuitability of these ligands for labelling the H2 receptor. We report here our results using 3H-tiotidine, a more potent H2-antagonist than either cimetidine or ranitidine, and show that this ligand meets the criteria for labeling the H2 receptor in guinea pig cerebral cortex membranes.

A species comparison of the histamine H2-receptor on mast cells and basophils.

The histamine H2-agonist dimaprit was employed in experiments to investigate the role of H2-receptors in mediator release systems from the rat, guinea-pig and man. Whereas inhibition of histamine release by dimaprit was observed in all 3 species, this effect appeared not always to be related to H2-receptor occupancy. Although the data in general support previous evidence for the presence of H2-receptors on human basophils and guinea-pig mast cells, the use of dimaprit as a pure H2-agonist in these studies is questioned. No evidence for H2-receptors on rat mast cells was obtained.

Further absorption studies with the anti-diarrhoeal agent ethacridine lactate in the dog.

2-Ethoxy-6,9-diaminoacridine lactate (ethacridine lactate, Rivanol, Metifex) has been administered orally to the dog once daily for 14 days, tritium labelled matterial having been given on days 1 and 14. The extent and rates of urinary excretion of radioactivity and the peak plasma levels and total radioactivity half-lives following the radiolabelled doses on days 1 and 14 were essentially the same. There was no significant change following multiple dosing in the level of urinary acridine-like material as determined fluorimetrically, which compared to approximately 0.01% of the dose found in the 0--24 h urine. It was concluded that, following oral administration of 3H-ethacridine lactate (5 mg/kg), less than 0.1% of the dose is absorbed as acridine-like material. Multiple dosing for 14 days does not alter this very low degree of oral absorption. In a separate study tritiated ethacridine lactate (30 microgram/kg) was administered i.v. to the dog. Approximately 84% of the radioactivity was eliminated in the 0--72 h post dose period, the majority of it being excreted via the faeces. There was a rapid loss of radioactivity from the plasma, followed by a long terminal phase in which acridine-like material was estimated to have a half-life of about 15 h.

Absorption studies with the anti-diarrhoeal agent ethacridine lactate in laboratory animals and man.

Tritiated 2-ethoxy-6,9-diaminoacridine (ethacridine lactate, Rivanol) has been orally administered to rat, dog, rabbit and man, and the distribution of radioactivity between urine and faeces determined. Complete recoveries of radioactivity were obtained from rat, rabbit and man. From direct measurement of levels of radioactivity, the maximum percentage of the dose excreted via the urine varied from 1.7% (man) to 6.1% (dog). Tritiated water accounted for nearly 50% of this amount. The low levels of radioactivity in plasma of dog and man and in the bile of rats following administration of 3H-ethacridine lactate suggested that there was only a low degree of oral absorption and substantiated previous reports that the drug is essentially unabsorbed in man. From fluorimetric measurements and the determination of levels of radioactivity in ether extracts of urine, it could be concluded that for all species less than 0.1% of the dose appeared in the urine as acridine-like material.